A new working hypothesis is formulated for the energy coupling to account for the different energy requirements for the shock-sensitive and resistant transport systems in E. coli. Experiments are proposed to test the validity of this hypothesis. These will take advantage of the unique property of a mutant of E. coli energy-uncoupled for active transport of amino acids and sugars which was isolated and studied in this laboratory, in its ease and completeness with which energy is uncoupled. Genetic experiments are proposed to dissect out the membrane energy transducing machinery. Biochemical characterization of the new Energy Coupling Factor (ECF) protein will be attempted. The role of cystathionase in active transport will be examined and the enzyme purified and characterized. Mitchell's chemiosmotic hypothesis for energy coupling of active transport will be examined using the energy uncoupled mutant, and the physiological role of carrier-mediated diffusion in transport will be assessed as well. The long-term goal of this research proposal is to broaden our knowledge concerning the molecular organization of the energy transducing machinery and the mechanisms of the coupling of metabolic energy to membrane functions, especially active transport, and to discover the membrane events that are necessary for bacteriocin, colicin K, to effect its lethal action, killing. It is anticipated that the results will be applicable to the more complex mammalian membrane systems.